The target of Rapamycin (TOR) is a highly conserved protein kinase found in both prokaryotes and eukaryotes. TOR proteins are members of the phosphoinositide (PI) 3-kinase related kinase (PIKK) family, which includes mammalian ATM, ATR, and DNA-PK (Tibbetts, and Abraham, Signaling Networks and Cell Cycle Control: The Molecular Basis of Cancer and Other Diseases, 5:267 (2000)). Like other PIKK family members; the TORs are large polypeptides (280-300 kDa) that bear a carboxy-terminal region with sequence similarity to the catalytic domains of PI3-kinases (PI3K) (Abraham et al., Annu. Rev. Immunol. 14: 483 (1996)). PIKK family members possessing active kinase domains phosphorylate proteins on serine or threonine residues. The consensus phosphorylation site for all PIKK family members (except the TORs) is serine/threonine followed by glutamine at the +1 position. The preferred sequence motif for the TORs remains unclear; however, known in vitro substrates contain serine/threonine followed by proline or a hydrophobic amino acid at the +1 position. The lack of a consensus motif for substrate recognition by TOR kinases hints that these PIKK family members may rely on an alternative mechanism for substrate identification or regulation in intact cells. (Abraham, Cell 111: 9 (2002)).
Several lines of evidence suggest that the TOR proteins function in a nutrient-sensing checkpoint control capacity. Both TOR and PI3K signaling are required for the activation (or inactivation) of several downstream effector proteins. However, whether TOR activity is regulated by PI3K, or whether the two signaling pathways function independently, is unknown. Overexpression of a membrane-targeted Akt/PKB protein (a downstream effector of PI3K) in mammalian cells leads only to a modest increase (or no change) in TOR kinase activity (as assayed in vitro), and moderately increases TOR autophosphorylation in vivo, as assessed with the S2481 phospho-specific antibody (Scott, et al, PNAS 95: 7772 (1998); Peterson, et al., J. Biol. Chem. 275: 7416 (2000); Sekulic, et al., Cancer Res. 60: 3504 (2000)).
Rapamycin is a bacterial macrolide with antifungal and immunosuppressant activities (Dumont et al., J. Immunol. 144: 251 (1990)). For example, rapamycin forms a complex with the immunophillin FKBP12 which then inhibits the kinase activity of TOR (Brown et al., Nature 369: 756 (1994); Kunz et al., Cell 73: 585 (1993)). Rapamycin treatment of cells leads to the dephosphorylation and inactivation of the cell-growth-promoting P70 S6 Kinase (Jeffries et al., EMBO J. 15: 3693 (1997); Beretta et al., EMBO J. 15: 658 (1996); Raught et al., PNAS 98: 7037 (2001)). In addition to rapamycin, wortmannin and LY294002 have also been shown to affect TOR signaling. Wortmannin is an active site inhibitor that abolishes TOR autophosphorylation. LY294002 blocks PI3K dependant Akt phosphorylation and kinase activity and inhibits TOR autophosphorylation as well. (Brunn et al., EMBO J. 15: 5256 (1996)). Rapamycin, wortmannin and LY294002 are commercially available from Cell Signaling Technology, Beverly, Mass., as Cat. Nos. 9904, 9951 and 9901, respectively.
Inactivation of the TOR proteins, or rapamycin treatment, mimics nutrient deprivation in yeast, Drosophila, and mammalian cells (Zhang, et al., Genes Dev. 14: 2712 (2000); Sekulic, et al., Cancer Res. 60: 3504 (2000); Nave, et al., Biochem. J. 344: 427 (1999); Barbet, et al., Mol. Biol. Cell 7: 25 (1996)). Thus, a current working model for TOR signaling proposes that these kinases relay a permissive signal to downstream targets only in the presence of sufficient nutrients to fuel protein synthesis. Because the TOR proteins appear to function in a coregulatory capacity with other conventional, linear signaling pathways, a passive nutrient sufficiency signal may be combined with stimulatory signaling from a second pathway to coordinate cellular processes that require the uptake of nutrients. The absence of either signal is predicted to prohibit activation of downstream targets. (Raught, et al, PNAS 98: 7037 (2001)).
It is believed that TOR signaling is affected through a combination of repression of phosphatase activity, and direct phosphorylation of downstream targets. Genetic screening in Saccharomyces cerevisiae has identified a phosphatase-associated protein (Tap42p), as one component of a rapamycin-sensitive signaling pathway (Schmelzle, et al., Cell 103: 253 (2000); Di Como, et al., Genes Dev. 10: 1904 (1996)). Tap42p phosphorylation is modulated by TOR signaling, which in turn regulates Tap42p interaction with phosphatases, such as PP2A (Jiang, et al., EMBO J. 18: 2782 (1999); Schmidt, et al., EMBO J. 17: 6924 (1998); Beck, et al., Nature (London) 402: 689 (1999); Di Como, et al., Genes Dev. 10: 1904 (1996); Jiang, et al., EMBO J. 18: 2782 (1999)). Tap42 orthologs are found in Arabidopsis (Harris, et al., Plant Physiol. 121: 609 (1999)), Drosophila, (GenBank accession number AAF53289), and mammalian cells (Inui, et al., J. Immunol. 154: 2714 (1995); Onda, et al., Genomics 46: 373 (1997)). In mammals, the B cell receptor binding protein α4 (a.k.a Ig binding protein 1, IGBP1) is the ortholog of Tap42p (Inui, et al., J. Immunol. 154: 2714 (1995); Onda, et al., Genomics 46: 373 (1997)).
TOR acts directly on other downstream targets, regulating the balance between protein synthesis and protein degradation in response to nutrient quality and quantity. TOR proteins regulate: (i) initiation and elongation phases of translation; (ii) ribosome biosynthesis; (iii) amino acid import; (iv) the transcription of numerous enzymes involved in multiple metabolic pathways; (v) autophagy, and (vi) expression of hypoxia-induced factor (HIF)-1 in oxygen-deprived cells (for a review see; Raught, et al., PNAS 98: 7037 (2001); see also, Hudson et al., Mol. Cell Bio.l 22: 7004 (2002)).
TOR signaling, in combination with the PI3K pathway, initiates the translation of rapamycin-sensitive mRNAs. In the presence of sufficient nutrients to fuel protein synthesis, TOR and PI3K signaling activate the S6Ks, and one or more unknown kinases, to effect phosphorylation of the ribosomal S6 protein, eIF4B, eIF4GI, and the 4E-BPs. TOR signaling has been reported to inhibit eEF2 phosphorylation (possibly via inhibition of the eEF2 kinase), thus, increasing elongation rates (Raught, et al., PNAS 98: 7037 (2001)).
Inhibition of TOR activity in S. cerevisiae potently represses translation initiation, concomitant with polysome disaggregation and cell cycle arrest in G.sub.1 (Barbet, et al., Mol. Biol. Cell 7: 25 (1996)). The mechanism for this translational repression is not understood, but could be due, at least in some strains, to the degradation of the initiation factor eIF4G (Berset, et al., Proc. Natl. Acad. Sci. USA 95: 4264 (1998); Powers, et al., Mol. Biol. Cell 10: 987 (1999)).
In addition to its effect on the phosphorylation state of proteins involved in translational control, TOR signaling regulates an abundance of the components of the translation machinery, at both the transcriptional and translational levels. Through the S6Ks, TOR signaling regulates the translation of ribosomal protein mRNAs in mammalian cells (Leicht, et al., Cell Growth Differ. 7: 1199 (1996); Mahajan, Int. J. Immunopharmacol. 16: 711 (1994)). Transfer of cultured mammalian cells from standard growth medium into amino acid and/or glucose-free medium leads to rapid dephosphorylation of two TOR substrates, S6K1 (p70 S6 kinase) and 4EBP1 (PHAS-I) (Gingras et al., Genes Dev 15: 807 (2001)). In the dephosphorylated state, S6K1 activity is repressed in starved cells, and nutrient stimulation leads to its phosphorylation and activation.
Activated S6K1 stimulates ribosome biogenesis, upregulating the translational capacity of the cell. Similarly, 4E-BP1 binds avidly to eIF-4E, thereby suppressing cap-dependent protein synthesis. Restoration of nutrients provokes multisite phosphorylation of 4E-BP1 by TOR (and possibly other protein kinases), release of eIF4E, and resumption of cap-dependent translation. Activators of TOR could also be useful in the therapy of muscle degeneration induced by diseases such as cancer or muscular dystrophy (for review, see Glass, Nature Cell Biol. 5(2):87 (2003)). Thus TOR is a central component of a rapamycin-sensitive signaling pathway that coordinates protein synthesis with glucose and amino acid availability. (Abraham, Cell 111: 9 (2002)).
In both yeast and mammalian cells, TOR signaling regulates autophagy. When nutrient levels are low, eukaryotic cells degrade cytoplasmic proteins and organelles to scavenge amino acids, in a process termed autophagy (Klionsky, et al., Science 290: 1717 (2000); Kim, et al., Annu. Rev. Biochem. 69: 303 (2000); Klionsky, et al., Annu. Rev. Cell Dev. Biol. 15: 1 (1999)). Switching yeast cells to a poor carbon or nitrogen source induces a state of quiescence (G.sub.0). Similarly, rapamycin addition to yeast cultures or to mammalian cells in culture induces autophagy, even in a nutrient-rich medium (Noda, et al., J. Biol. Chem. 273: 3963 (1998); Blommaart, et al., J. Biol. Chem. 270: 2320 (1995)). TOR signaling modulates gene expression via cytoplasmic sequestration of several nutrient-responsive transcription factors. TOR signals to several specific effectors (Tap42, Mks1p, Ure2p, Gln3p, and Gat1p) eliciting changes in the expression levels of enzymes involved in several different metabolic pathways (Shamji, et al., Curr. Biol. 10: 1574 (2000); Chan, et al., PNAS USA 97:13227 (2000)). How TOR signaling may affect the transcription rates of metabolic enzymes in multicellular organisms has not yet been elucidated (Blommaart, et al, J. Biol. Chem. 270: 2320 (1995); Shigemitsu, et al., J. Biol. Chem. 274: 1058 (1999)).
In mammalian cells, autophagy is inhibited by amino acids and insulin. Activation of S6K is associated with inhibition of autophagy in rat hepatocytes, and the inhibition of autophagy by amino acids could be partially prevented by rapamycin treatment (Blommaart, et al., J. Biol. Chem. 270: 2320 (1995); Shigemitsu, et al, J. Biol. Chem. 274: 1058 (1999)).
The observation that rapamycin can inhibit long-term facilitation in Aplysia neurons has implicated TOR signaling in the control of neuronal protein synthesis (Casadio, et al, Cell 99: 221 (1999)). Several types of neurotransmitters were described to affect the activity of the rapamycin-sensitive pathway leading to S6K1 and 4E-BP1 phosphorylation. Serotonin (5-HT) addition to Aplysia neurons or Chinese hamster ovary (CHO) cells expressing the 5-HT1B receptor increases phosphorylation of S6K1 in a rapamycin-dependent manner (Khan, et al., Neurosci. 21: 382 (2001); Pullarkat, et al., J. Neurochem. 71: 1059 (1998)). Dopamine addition to CHO cells also activates S6K1 in a rapamycin-dependent manner (Welsh, et al., J. Neurochem. 70: 2139 (1998)). Finally, both S6K1 and 4E-BP1 phosphorylation is induced by stimulation of the μ-opioid receptors (which mediate the analgesic and addictive properties of morphine) by the agonist [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO; Polakiewicz, et al., J. Biol. Chem. 273: 23534 (1998)). TOR interacts with gephyrin, a tubulin-binding protein involved in neuronal γ-aminobutyric acid type A (GABAA) and glycine receptor clustering. (Sabatini, et al., Science 284:1161 (1999); Scheper, et al., J. Biol. Chem. 267: 7269 (1992); Essrich, et al., Nat. Neurosci. 1: 563 (1998); Kirsch, et al., Nature (London) 366: 745(1993)). Gephyrin binding is reportedly required for signaling to S6K1 and 4E-BP1, and, consistent with a role in localized protein synthesis, a fractionation experiment demonstrated that TOR and gephyrin were enriched in the synaptosomal fraction but not in the post synaptosomal fraction (Sabatini, et al., Science 284: 1161 (1999)).
The role of TOR in the numerous signaling pathways listed above, as well as others, makes modulating TOR activity a potentially desirable treatment method for a variety of conditions. For example, TOR kinase activity is important for antigen-activated T-cell proliferation, thus, rapamycin, which inhibits TOR activity, is useful as an immunosuppressant. (Abraham et al., Annu. Rev. Immuno. 14: 483 (1996); Morice et al., J. Biol Chem 268: 3734 (1993)). Similarly, TOR has been identified as a therapeutic target involved in human cancers (Huang et al., Current Opinion in Investigational Drugs. 3(2):295 (2002)). However, there is a need in the art to fully understand the role of TOR in many signaling pathways, and to identify the various modulators of TOR kinase activity within these pathways. Such modulators, once identified, may be useful in treating many of the disorders associated with TOR signaling pathways.
A few TOR modulators, such as rapamycin, wortmannin and LY29004 have already been identified; however, the discovery of additional modulating agents is necessary to address the numerous conditions affected by TOR. However, current assays for screening for such agents rely on recombinant TOR, and produce, at best, poor results. For example, in mammalian cell lines, recombinant TOR yields are too low to be useful in any assay. Similarly, in bacterial cell lines, recombinant TOR is expressed in an insoluble form, and thus cannot be used in an assay. In addition, recombinant TOR, when expressed in insect cell lines, has no detectible kinase activity, and thus is not useful in any assays. Such limitations serve as a barrier to elucidating TOR's role in many signaling pathways and in identifying TOR modulators, which may be useful in treating the many disorders associated with TOR signaling. Thus, because of the limitations of recombinant TOR, there are no useful assays in the current art employing recombinant TOR. This is particularly true for high throughput screening assays, which are even less tolerant of recombinant TOR's limitations. Accordingly, there is a need in the current art for a TOR assay that measures TOR kinase activity, and that is adaptable to high throughput screening.